A and antioxidants hence protects cells against the

A decrease in the CAT activity in MTX-treated
cells compared to that of the untreated cells has been observed. MTX ratchets down the activity of the CAT as an antioxidant enzyme (Çetin et al., 2008; Chang et al., 2013). In our experiment a significant increase
in the CAT activity in TAT-CPG2
pretreated cells has been observed. Therefore, transduced TAT-CPG2 prevents the
accumulation of MTX inside the cells and maintains the balance between oxidants
and antioxidants hence protects cells against the oxidative stress induced by

Based on the in vitro results, and reported mechanisms
for MTX cell toxicity, also considering HepG2 as a proliferating cell line, one might conclude that in the cell death induced by MTX in HepG2 cells both mechanisms
of cell cycle suppression caused by the inhibition of dihydrofolate
reductase and
oxidative stress caused by the accumulation of MTX are involved.
Therefore, transduced TAT-CPG2 converts
MTX into its non-toxic metabolites and prevents the accumulation of MTX in the cell and thus its
toxic effect.

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Delivery of
CPG2 into the cells by protein transduction is potentially valuable for a strategy known as enzyme/prodrug therapy. CPG2 is able to hydrolyze
specifically the amido, carbonyl or ureido bonds between
L-glutamic acid and the carboxyl-, phenol or aniline-substituted aromatic
rings, respectively (1,2). Several prodrugs such as
4-(2-chloroethyl)(2-mesyloxyethyl)amino-benzoyl-L-glutamic acid (CMDA) and
ZD2767P have been synthesized to release potent DNA-alkylating
mustard drugs. These prodrugs are utilized in CPG2-medited strategies, such as antibody- or gene-directed enzyme prodrug therapy (ADEPT
or GDEPT) (Jamin et al.,
2011; Capucha et al., 2012; Karjoo et al., 2016). Because of disadvantages
of cancer gene therapy such as safety problems, it has
been proposed that direct delivery of proteins into the cell is an alternative to gene therapy, especially gene therapy of those type of cancers that do not require long-term sustained and regulated
expression of the transgene (Ford et al., 2001). Therefore, we will propose that
TAT-CPG2 fusion protein can be used as an alternative to the GDEPT approach.



We have shown the construction, expression and
purification of CPG2 fused to HIV-1 TAT peptide (TAT–CPG2) in this study. We
have demonstrated for the first time that TAT-CPG2 in both native and denatured
forms can be efficiently transduced into the HepG2 cells. Also, we have provided
evidences for the enzyme activity of transduced TAT-CPG2 fusion protein.
Furthermore, we have shown that TAT-CPG2 fusion protein strongly protects HepG2 cells against MTX-induced cell death.
We assume that transduced CPG2 converts MTX to non-toxic metabolites and
prevents the accumulation of MTX in cells and therefore prevents the cell proliferation suppression and oxidative stress caused by
MTX. However, further studies
are required to elucidate the involved cellular mechanisms. Our success in the protein transduction of TAT-CPG2 may provide a new strategy for
protecting against cell toxicity resulting from MTX in various organs and
also may provide an opportunity for the development of therapeutic methods for
the treatment of cancer by enzyme/prodrug strategy.


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